Actin is one of the most abundant human proteins, but very little understanding exists about why actin is so highly conserved. Over 300 million years of evolution there have been no amino acid changes in the skeletal striated muscle isoform of actin. Similarly, there is no understanding of why most sequence differences are between tissue-specific isoforms and not between species. The hypothesis that is being investigated in this research is that many allosteric linkages exist within the actin filament, placing constraints and selective pressure on all of the buried residues. These allosteric linkages are essential for the many functions of actin, from muscle contraction to cell motility to forming the stereocilia responsible for hearing. Past productivity has been strong in this project, but the proposed period represents a major turning point. A new state-of-the-art electron microscope and improved image analysis methods will allow most of the proposed aims to be accomplished at high resolution where near-atomic details of the interactions within the actin filament and between actin and actin-binding proteins can be visualized. The proposed aims include studies of different isoforms of actin, the role of actin-nucleating proteins in determining the structure and structural dynamics of an actin filament, and many complexes of actin filaments with other proteins. Some of these actin-binding proteins contain structurally conserved domains, such as the Calponin Homology (CH) domain or Immunoglobulin (Ig) domains, and the hypothesis is that the presence of such domains tells us only about the ancestry and evolution of these proteins and not about how these domains interact with actin. Thus, different CH domains may interact in a completely different manner with actin, or not interact at all, as was shown for the CH domain within the eponymous protein calponin. The proposed studies will be done in collaboration with other laboratories using x-ray crystallography, NMR, biochemistry and mutagenesis, and the resulting work should have a large impact on our understanding of how actin functions and interacts with many other proteins. PUBLIC HEALTH RELEVANCE: Actin is a protein important to cellular processes from muscle contraction to the metastasis of malignant cells. Our research is aimed at understanding how actin functions, how mutations in actin cause human diseases, and how pathogens target actin as part of bacterial infections.